Emerging studies strongly support that post-traumatic stress
disorder (PTSD) secondary to combat exposure leads to an increased risk
for early morbidity and mortality. It is becoming evident that
psychological stress is associated with oxidative stress, including
increased pro-inflammatory cytokines and peripheral markers of
oxidative stress [1]. Various cytokines were investigated in studies
related to Veterans world-wide suggesting that PTSD confers a
pro-inflammatory state [2]. Recent studies have examined the complex
interplay between the neuroendocrine and immunological changes in PTSD
and showed that PTSD is associated with a pattern of poor physical
health outcomes and a risk of illness in later life that is consistent
with altered inflammatory responsiveness [3].

Major depressive disorders have been
associated with high activity of acid sphingomyelinase (ASMase), a
lipid metabolizing enzyme that hydrolyses sphingomyelin (SM) into
ceramide and phosphorylcholine, and can be inhibited with tricyclic
antidepressants (TCA) [4]. Activation of ASMase can lead to
interleukin-1 (IL-1) release from brain astrocytes [5], which in turn
activates the hypothalamic-pituitary-adrenal axis and elevates plasma
corticosterone levels [6]. Increased activity of ASMase has also been
implicated in the pathophysiology of common diseases including
cardiovascular disease (CVD) [7].

To determine whether higher
pro-inflammatory cytokine levels and increased CVD risk in PTSD could
be correlated, we analyzed inflammatory markers and secretory ASMase
(S-ASMase) activity in the plasma of both combat Veterans with PTSD and
healthy individuals. While the Veterans taking statins displayed lower
levels of total cholesterol, LDL cholesterol and non-HDL cholesterol;
levels of pro-inflammatory cytokines were elevated compared to non-PTSD
controls. The anti-inflammatory effects of statins have been described
[8]; however, how statins influence HDL functionality and whether HDL
retains pro- or anti-inflammatory properties are still obscure [9]. In
addition, PTSD patients in the current study exhibited increased
activity of the secreted form of ASMase (S-ASMase) in the plasma, and
had elevated plasma levels of the pro-inflammatory sphingolipid
sphingosine 1-phosphate (S1P). Our findings suggest that combat
Veterans with PTSD, despite being on statin treatment, still exhibit
high CVD risk factors in the form of elevated plasma levels of the
pro-inflammatory cytokines and the sphingolipid S1P.

MATERIALS AND METHODS

Participants

This pilot study was approved by the institutional
review board at the Medical University of South Carolina (MUSC), and
proper consent was obtained from each subject. Eight combat Veterans
were diagnosed with PTSD using the Clinician Administered PTSD Scale –
Diagnosis (CAPS-DX), and co-morbidity was assessed with the
Mini-International Neuropsychiatric Interview [10-11]. A CAPS score of at
least 50 and PTSD symptoms of at least moderate severity on the
Clinical Global Impressions Scale [12] were used in the study. Blood
samples were collected in Monoject™ lavender stopper collection tubes
(#8881311743) (COVIDIEN, Mansfield, MA) with
ethylene-diaminetetraacetic acid (EDTA) as anticoagulant. After plasma
samples were separated, they were aliquoted into 0.5-ml aliquots and
stored at –80ºC until processed.

The control group consisted of five
male subjects who were screened for healthy levels of conventional
lipid panel (total cholesterol, HDL-cholesterol, LDL-cholesterol,
VLDL-cholesterol, and triglycerides), glucose, C-reactive protein
(CRP), complete blood count, platelet count, and comprehensive
metabolic panel including liver and kidney function. A more detailed
summary of the clinical profile of the control group has been
previously published [13].

Plasma lipid profile analysis

Architect Immuno-chemistry analyzer (C16200, Abbot
Diagnostics) was used to analyze the plasma lipid profile for the PTSD
patients at the VA Clinical Laboratory at Ralph H. Johnson VA Medical
Center, and for the healthy controls at the clinical laboratories of
MUSC Medical University Hospital. Results were confirmed using the
Cholestec LDX® system (Cholestec Corporation, Hayward, CA) in our
laboratory.

ASMase activity was assayed on duplicate samples
using a slight modification of the protocol published by Jenkins et al. [14]. Briefly, S-ASMase activity was performed with 100 ml of human plasma and assayed with a reaction buffer containing 100 μm porcine brain sphingomyelin, 1×105 cpm of choline-[methyl-14C] in micelles containing 0.2% Triton X-100 in sodium acetate buffer (250 mm, pH 5.0) with 0.1 mm ZnCl2. The reaction was run for 60 min at 37°C, then terminated by the addition of 800 µl
of chloroform/ methanol (2:1, v/v) followed by the addition of 0.2 ml
of Milli-Q deionized water. After mixing and centrifugation at 2,000×g
for 5 min, the upper (aqueous) phase was removed and used for liquid
scintillation counting.

Statistical analysis

Data are presented as mean ± standard error of the
mean; p<0.05 was considered statistically significant. Comparisons
between lipid panels, sphingolipid profiles, and S-ASMase activities
were performed using two-tailed student’s t test. Comparisons between
cytokine profiles were performed using the Mann-Whitney rank sum test.

RESULTS

Effect of statin treatment in combat Veterans with
PTSD

Information related to age, diagnoses, and whether the PTSD
patient was treated for hyperlipidemia and depression at enrollment is
summarized in Table 1.

Table 1: Information related to age, diagnoses, and
whether the combat Veteran PTSD patient was treated (Rx) for hyperlipidemia and
depression at enrollment.

Patient

Age

Diagnosed condition

Statin Rx

PTSD Rx

1

60

Hypertension
(HTN)

NO

NO

2

59

Chronic
obstructive pulmonary disease +HTN

YES

YES

3

23

No cardiovascular disease symptoms

NO

NO

4

65

HTN + hyperlipidemia

YES

YES

5

64

HTN + hyperlipidemia

NO

YES

6

59

Hyperlipidemia

YES

YES

7

58

Hyperlipidemia
+ peri-pheral vascular disease

YES

YES

8

58

HTN + atherosclerosis

YES

YES

Plasma lipid profiles of the PTSD patients were measured before and after enrollment and statin treatment (Table 2).

Table 2:Plasma lipid profile analysis (mg/dl) of
Veterans with PTSD before and after treatment with statins (n=8). Data are
Mean±SE. TC, total cholesterol, TRG triglycerides

Patient

TC

HDL

TRG

LDL

Non-HDL

TC/HDL

Before Statin

207.29±14.5

42.71±3.9

190.00±31.5

126.43±16.9

164.57±15.1

5.11±0.6

After Statin

169.88±10.3

46.50±8.4

192.38±29.0

84.43±10.9

126.14±11.1

4.53±0.6

p values

0.052

0.703

0.957

0.063

0.069

0.520

While the average total cholesterol levels were over the recommended
maximum of 200mg/dl before treatment, after statin use this level
decreased to below 200mg/dl. Levels of LDL and non-HDL cholesterol were
reduced with statin treatment.

Plasma from
PTSD patients had significantly increased levels of the
pro-inflammatory cytokines IL-6, IL-10, IFN-γ, and TNF-α compared to
healthy controls (Figure 1A). While plasma sphingolipids were generally
higher in the PTSD group compared to the control group, of note was the
significantly higher levels of S1P (Figure 1B). Additionally, levels of
C18-ceramide and dihydro-S1P (dh-S1P) were also significantly higher in
the PTSD group. Sub-clinical inflammation may have contributed to
greater variability in levels of cytokines compared to sphingolipids.

Levels of S-ASMase activity were measured in the
plasma of the control and PTSD groups to determine whether there was a
correlation between increased S-ASMase activity and PTSD. The mean
activity of S-ASMase was significantly 60% higher (p=0.003) in the PTSD
group compared to the healthy group (Figure 2). Therefore, increased
S-ASMase activity in the plasma may be correlated with PTSD.

Figure 2.
Analysis of secretory ASMase (S-ASMase) activity in plasma samples from
Veterans with PTSD and healthy controls. Plasma (100 µl) was analyzed
for S-ASMase activity as described in Materials and Methods. Each data
point shown is a mean of duplicate determinations, group means are
significantly different at p=0.003

DISCUSSION

A major challenge in addressing the causal
relationship between PTSD and poor health is that existing studies on
PTSD have measured physical health using the traditional self-report
measures of physical health, which may be influenced by psychological
health. More reliable measures would involve factors that can be
analyzed through laboratory tests. The pro-inflammatory markers
measured in our study might be used to provide a diagnosis/prognosis of
PTSD and concomitant diseases such as cardiovascular, gastrointestinal,
and musculoskeletal disorders.

Despite widespread use of statins to
reduce CVD risk, there is considerable variability in the
cholesterol-lowering response among individuals [15, 16, 17]. The origin of
this variability is poorly understood; however, pharmacogenomic studies
have begun to uncover associations of candidate genes with alterations
in cholesterol-lowering response to statins [18, 19, 20] It appears that the
genetic contribution to the variability in statin-mediated cholesterol
reductions result from combined effects from multiple polymorphisms
with minimal contribution from individual genes [20, 21]. Using a targeted
lipidomics platform, Kaddurah-Daouk et al found no overlap of lipid
changes that correlate with LDL cholesterol or CRP responses to
simvastatin, which suggested that distinct metabolic pathways regulate
statin effects on the two biomarkers [22]. The sphingolipidomics approach
described in this study could in future large population studies
provide insights into responses to statins including
inflammation-related pathways.

Despite the apparent trend of statins
in lowering blood cholesterol observed in the PTSD group,
pro-inflammatory cytokines and S1P were still higher than in the
control group, suggesting other CVD risks such as chronic inflammation
remains. A pooled analysis of four studies measuring carotid
intimal-media thickness (cIMT) in randomized familial
hypercholesterolemia patients concluded that while the rate of cIMT
development slows considerably in response to intensive statin therapy,
the complications of atherosclerosis also depend on the degree of
inflammation and other physical properties of the artery [23].
Inflammatory markers such as CRP and soluble CD40 ligand have been
shown to decrease in response to aggressive statin therapy [24, 25];
however, studies on patients taking various lipid-lowering drugs have
shown that there remains a ‘residual risk’ in the development of CVD,
perhaps because underlying inflammatory causes were not
addressed [23, 26, 27, 28]. Therefore, it would appear that with statin use the
duration of treatment, the type of statin, and the patients studied are
influential in determining the outcome on CVD risk. Our data imply that
the PTSD group in this study may still be at higher risk for
development of CVD despite the statin treatment.

Numerous studies addressed the role
of cholesterol in the regulation of synaptic transmission; however, the
cellular mechanisms by which cholesterol deficiency mediates the
pathological events in the nervous system are poorly understood. It has
been suggested that the base line of the extracellular glutamate
concentration in the brain can have a major effect on the neuronal
excitability and synaptic transmission. Recently, it was demonstrated
that reduced levels of cholesterol in the plasma membrane of neurons
causes increase in ambient glutamate [29]. In case of PTSD the application
of statins may therefore exacerbate the clinical course of depression.

Atherosclerotic plaque buildup in the
vessel wall is typically initiated by activated macrophages
internalizing modified LDL particles. Activated macrophages release
cytokines, such as TNF-α, which can cause local inflammation and
recruit more macrophages to the area. We have previously reported that
S1P is able to induce increases in released TNF-α, and prostaglandin E2
in macrophages in vitro [30]. We have also reported that sphingosine
kinase, the enzyme that generates S1P, is released by human monocytic
cells in response to modified LDL immune complexes, generating
extracellular S1P that may be involved in sustained activation [30, 31].
Our current data suggest that PTSD can lead to increased
pro-inflammatory cytokines and S1P in the plasma that may drive the
pro-inflammatory mechanisms contributing to the development of vascular
disease; however, the significance of elevated dh-S1P levels in PTSD
patients is not clear.

It has been shown that S-ASMase can
hydrolyze SM in LDL particles causing an increase in particle size and
a tendency to aggregate and stick to the vessel wall [32]. This finding
suggests that the doses of ASMase-inhibiting effects of TCA drugs such
as Norpramin (desipramine) [4] could be optimized to further reduce plasma
S-ASMase activity to non-PTSD levels. Furthermore, it has been shown
that the hydrolysis of SM to ceramide by ASMases is a necessary step in
the pathway for the production of S1P [33]. Our current data show that
plasma ceramide levels were elevated in the PTSD patients, which
support the notion that this ceramide pool can be converted to S1P.

The relatively low number of patients
merit that caution must be used in interpreting the results. For
instance, despite lower levels of plasma total and LDL cholesterol
levels in PTSD patients after statin treatment, statistically
significant differences before and after statin treatment were not
attained. The expansion of this study to include more patients would
add more statistical power. In spite of the small sample size, the
results of this pilot study brings up the interesting possibility of
whether chronic inflammatory processes are causally involved in major
depressive disorders and it is intriguing to hypothesize that lowering
the pro-inflammatory symptoms may also contribute to the treatment of
major depressive disorders such as PTSD.

ACKNOWLEDGEMENTS

This work was supported by NIH Grants HL-079274 and HL-079274-04S1
(ARRA), The Southeastern Clinical and Translational Research Institute
(SCTR, formerly GCRC) and the South Carolina Center of Biomedical
Research Excellence (COBRE) in Lipidomics and Pathobiology (P20
RR17677) to S.M.H; and funding from the Medical Research Service, Ralph
H. Johnson VAMC, Charleston, to M.B.H. Special thanks to the Lipidomics
Shared Resource facility at MUSC for sphingolipid analysis, and for the
MUSC Proteogenomics Facility for the use of the Bioplex system
for cytokine determination.

COMPETING FINANCIAL INTERESTS

MBH has shares in Merck, and has been awarded grants from Pfizer Inc. and Otsuka Pharmaceuticals.